The human body needs protein for growth and maintenance. Aside from water, protein is the most abundant molecule in the body. According to U.S. and Canadian Dietary Reference Intake guidelines, women aged 19-70 need to consume 46 grams of protein per day, while men aged 19-70 need to consume 56 grams of protein per day to avoid deficiency. This recommendation, however, is for a sedentary person free of disease. Protein deficiency can lead to reduced intelligence or mental retardation as well as contribute to the prevalence of diseases such as kwashiorkor. Protein deficiency is a serious problem in developing countries, particularly, in countries affected by war, famine, and overpopulation. Animal sources of protein, such as meat, are often a source of the complete complement of all the essential amino acids in adequate proportions.
The nutritional benefits of meat are tempered by potential associated environmental degradation. According to a 2006 report by the Livestock, Environment And Development Initiative, entitled Livestock's Long Shadow—Environmental Issues and Options, the livestock industry is one of the largest contributors to environmental degradation worldwide, and modern practices of raising animals for food contributes widely to air and water pollution, land degradation, climate change, and loss of biodiversity. The production and consumption of meat and other animal sources of protein is also associated with the clearing of rainforests and species extinction. Accordingly, there is a need for a solution to demands for alternative to meat produced from live animals.
The inventors have previously described engineered meats and methods of making engineered meats using cultured cells. See, e.g., U.S. Pat. No. 8,703,216, titled “ENGINEERED COMESTIBLE MEAT,” previously incorporated by reference in its entirety. However, bio-manufacturing processes aimed at building extended tissue constructs that require large numbers of adherent cells face the difficulty of growing these cells (to the billions to trillions) efficiently and cost effectively. One way to produce large quantities of such cells is to use microcarriers in bioreactor-based systems. Unfortunately, commercially available microcarriers are not appropriate for use in a comestible meat product, as the carrier is not typically edible or fit for consumption. Such commercially available microcarriers are typically composed of natural products (e.g., cross-linked dextran, collagen, alginate) or synthetic materials (glass, polystyrene, acrylamide). Typically, commercially available microcarriers are not edible, since they are made of synthetic materials. Further, commercially available microcarriers are not made from animal-product-free (e.g., they are made from animal products), and/or may be made from bacterial products (e.g., collagen, dextran) which raise issues of contamination or allergy making them unsuitable for eating. Thus, such microcarriers may have to be separated from the cultured cells in order to form a viable meat product. For example, existing or proposed microcarriers may be composed either of synthetic polymers (Polystyrene, poly(L-lactide), poly(N-isopropylacrylamide, PLGA) or of animal (or bacterial) derived polymers (e.g., gelatin, recombinant gelatin, dextran). None of these microcarriers would fit our need because they are not edible (synthetic) or safe for consumption (e.g., dextran, recombinant gelatin) with a substantial risk of bacterial contaminant and/or they require killing animals (e.g., gelatin).
In general, microcarriers may increase the useful surface-area-to-volume ratio considerably compared to 2D cell culture systems. However, because harvesting healthy cells separated from the microcarriers can be challenging, such approaches are used mainly in applications where the cells are not the final product such as virus and metabolite production. See, e.g., U.S. Pat. No. 7,270,829, which describes the use of microcarriers such as Cytodex 1 and 3 beads and the use of EDTA, trypsin, and centrifugation to remove the microcarriers from the cultured cells.
A developing field within biofabrication/biomanufacturing aims at producing animal products for human consumption (i.e., cultured meat) without killing animals. As such it shares the challenge of producing large quantities of cells and raises other challenges for microcarriers. Thus, it would be beneficial to develop a new type of microcarrier with characteristics that may include: (1) the microcarrier should be edible and digestible so they can be incorporated into the final comestible product (e.g., no synthetic material, no toxic chemical used for their formation); (2) the microcarriers need to have been formed of an animal-free composition to assure that the final product retains its no animal kill character (e.g., no collagen, gelatin, etc.); (3) production of the microcarriers needs to be scalable and low cost; and (4) the microcarriers may bring additional features to the final product (e.g., gustatory benefit, mouthful feeling, health benefits i.e., higher content of fibers, etc.).
Described herein are edible microcarriers that may meet some or all of these criteria and may be used to form comestible meat products using cell culture methods, as well as methods of making the, methods of making engineered meat products with them, and the resulting engineered meat.
While microcarriers for cell culture have been described before, previously described microcarriers are typically used as substrates for cell culture and are not part of the final product (i.e. cells or cell secreted proteins). Further, these technologies are often designed as injectable formulations for therapeutic applications. The use of edible microcarriers composed entirely of animal-product-free (“animal-free”) molecules for cell culture is a novel platform technology for meat production.